Synthesis, Biological Evaluation, And Molecular Modeling of Chalcone

Article pubs.acs.org/jmc

Synthesis, Biological Evaluation, And Molecular Modeling of Chalcone Derivatives As Potent Inhibitors of Mycobacterium tuberculosis Protein Tyrosine Phosphatases (PtpA and PtpB) Louise Domeneghini Chiaradia,†,‡ Priscila Graziela Alves Martins,† Marlon Norberto Sechini Cordeiro,‡ Rafael Victorio Carvalho Guido,§ Gabriela Ecco,† Adriano Defini Andricopulo,§ Rosendo Augusto Yunes,‡ Javier Vernal,† Ricardo José Nunes,*,‡ and Hernán Terenzi*,† †

Centro de Biologia Molecular Estrutural, CEBIME−UFSC, Universidade Federal de Santa Catarina, Campus Trindade, 88040−900 Florianópolis−SC, Brasil ‡ Laboratório Estrutura e Atividade, Departamento de Química, LEAT−CFM−UFSC, Universidade Federal de Santa Catarina, Campus Trindade, 88040−900 Florianópolis−SC, Brasil § Laboratório de Química Medicinal e Computacional, Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador São-Carlense 400, 13560−970 São Carlos−SP, Brasil. S Supporting Information *

ABSTRACT: Tuberculosis (TB) is a major infectious disease caused by Mycobacterium tuberculosis (Mtb). According to the World Health Organization (WHO), about 1.8 million people die from TB and 10 million new cases are recorded each year. Recently, a new series of naphthylchalcones has been identified as inhibitors of Mtb protein tyrosine phosphatases (PTPs). In this work, 100 chalcones were designed, synthesized, and investigated for their inhibitory properties against MtbPtps. Structure−activity relationships (SAR) were developed, leading to the discovery of new potent inhibitors with IC50 values in the low-micromolar range. Kinetic studies revealed competitive inhibition and high selectivity toward the Mtb enzymes. Molecular modeling investigations were carried out with the aim of revealing the most relevant structural requirements underlying the binding affinity and selectivity of this series of inhibitors as potential anti-TB drugs.

1. INTRODUCTION Tuberculosis (TB) is an important disease due to infection by Mycobacterium tuberculosis (Mtb) which primarily affects the respiratory tract. According to the World Health Organization (WHO), there are nearly 10 million new cases of TB and 1.8 million deaths each year. The situation is further worsened by the co-infection with HIV due to the devastating effects of Mtb on the vulnerable immune system of the patients.1 Despite BCG vaccine (Bacillus Calmette−Guerin) and the combined chemotherapy with first-line (isoniazid, ethambutol, rifampicin, pyrazinamide) or second-line (ethionamide) antibiotics (WHO), Mtb is the agent which causes more deaths from infection in the world. The chemotherapy involves the use of these four antibiotics for 6−9 months, resulting in significant lack of patient adherence to long-term therapy. The global emergence of multidrug-resistant (MDR) strains of Mtb is also a serious health problem.2 For these reasons, there is an urgent need for new safe and effective anti-TB drugs. The protein tyrosine-phosphatases (PTPs) are a family of regulatory enzymes that are closely related, and together with © 2011 American Chemical Society

protein tyrosine kinases (Ptks) have a key role in the control of the phosphorylation state of tyrosines in the cells. The interaction between these two classes of enzymes is crucial to control the phosphorylation/dephosphorylation cascades, which are involved in several cellular processes.3 On the basis of the Mtb genome, it was possible to identify two phosphotyrosine phosphatase genes, MtbPtpA and MtbPtpB.4 While PtpA is classified as a low-molecular-weight protein (LMW PTP),5 which specifically dephosphorylates tyrosine residues,6 PtpB is a phosphatase with triple specificity (TSP PTP), which catalyzes the dephosphorylation of both residues (serine/threonine or tyrosine) and also has phosphoinositide phosphatase activity.7,8 The enzymes PtpA and PtpB are secreted by Mtb into the host cell and attenuate host immune defenses by interfering with the host signaling pathways.9−11 Confirming this fact, it was identified in macrophages the vacuolar protein sorting 33 homologue B (VPS33B), a Received: September 20, 2011 Published: December 2, 2011 390

dx.doi.org/10.1021/jm2012062 | J. Med. Chem. 2012, 55, 390−402

Journal of Medicinal Chemistry

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Scheme 1. Synthesis of Chalcone Derivatives 1−47a

a

(a) KOH 50% w/v, methanol, rt, 24 h. *Novel compounds. Ph = phenyl.

by targeting the IFN-γ mediated signaling pathway.15 These findings elegantly demonstrate that MtbPtpB subverts the innate immune responses by blocking extracellular signalregulated kinases 1 and 2 (ERK1/2) and p-38 mediated by IFN-γ stimulated interleukin 6 (IL-6) production and promotes host cell survival by activating the Akt pathway and blocking caspase-3 activity.15 Moreover, Bach and co-workers12 and Zhou and co-workers15 demonstrated that PtpA and PtpB are essential for intracellular bacterial persistence, suggesting that the inactivation of these enzymes reduces the growth of Mtb in human macrophages. Because of the significant regulatory role in the cell signaling, dysfunctions of activity in the human PTPs provoke diseases such as cancer, diabetes, neurologic, and autoimmune disorders. For these reasons, PTPs appear as important therapeutic targets.16−18 In line with this, the specific inhibition of MtbPtpA and MtbPtpB emerges as a highly promising new approach for TB therapy.15,18,19

regulator of membrane fusion, as a MtbPtpA substrate. PtpA enters into host macrophages and dephosphorylates the cytoplasmic protein VPS33B, inhibiting the maturation of phagosomes, and therefore, preventing their fusion with lysosomes, a mechanism by which macrophages promote their microbicidal activity.12 Hence, PtpA was recognized as the first enzyme of Mtb that interacts directly with an identified substrate of the host. Due to the fact that PTPs of Mtb modulate phosphorylated proteins of the host, it was believed that Mtb lacked tyrosine kinases proteins.4 However, it was recently identified in Mtb a protein tyrosine kinase (PtkA), which has PtpA as substrate, although PtkA is not a substrate for PtpA.13 Regarding MtbPtpB, its genetic inactivation accelerated mycobacterial cell death in interferon-γ (IFN-γ) activated macrophages and severely reduced the bacterial load in guinea pigs.14 Recently, Zhou and co-workers hypothesized that MtbPtpB could promote mycobacterial survival in the host 391



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Scheme 2. Synthesis of Chalcone Derivatives 48−100a

a

(a) KOH 50% w/v, methanol, rt, 24 h. *Novel compounds. Ph = phenyl.

The pioneers in the description of MtbPtpA inhibitors, Waldmann and co-workers, have tested analogues of the natural products stevastelin, roseofilin, and prodigiosins, obtaining IC50 values ranging from 8.8 to 28.7 μM.20 Later, sodium molybdate, orthovanadate, and tungstate were characterized as reversible inhibitors of PtpA.21 Ellman and co-workers also identified a benzanilide derivative as selective inhibitor of PtpA, with a Ki value of 1.4 μM.22 In the search for MtbPtpB inhibitors, Alber and co-workers studied the sulfonamide derivative (oxalylamino-methylene)thiophene sulfonamide (OMTS), which showed high specificity and selectivity for this protein in comparison with other human PTPs, with an IC50 value of 0.44 μM.23 They also identified a series of indole derivatives as PtpB inhibitors, showing selectivity indexes up to about 100.24 Isoxazoles were found as competitive inhibitors of PtpB, with the most selective inhibitor presenting high affinity with a Ki value of 0.22 μM.25

More recently, thiazolidinones spiro-fused to indolinones and sulfonylhidrazones were also identified as MtbPtpB inhibitors.26 In recent years, we have described the inhibitory potency of a series of naphthylchalcones against MtbPtpA, with the most potent compound exhibiting an IC50 value of 8.4 μM.27 These are competitive and selective inhibitors of MtbPtpA, which additionally showed significant growth inhibitory activity of Mtb in infected macrophages.28 Yoon and co-workers also identified licochalcone A derivatives as inhibitors of PTPs (human PTP1B).29 Similarly, many classes of compounds were assayed in cultures of Mtb,30 and only three studies were conducted with chalcones;31−33 nevertheless, no information about the molecular target of these compounds was reported. Therefore, the interest in this class of compounds has grown rapidly not only by its general chemistry and biological properties but also by the need for new anti-TB drugs. As part of our research program aimed at developing new lead candidates for TB therapy,27,28 we have selected chalcones, 392



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Table 1. Yields of the Synthesis and Percentage of Inhibition of MtbPtpA and MtbPtpB at a Single Concentration of 25 μMa

a

compd

yield (%)

MtbPtpA inhibition (%)

MtbPtpB inhibition (%)

compd

yield (%)

MtbPtpA inhibition (%)

MtbPtpB inhibition (%)

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81 83 85 87 89 91 93 95 97 99

93 90 98 93 88 92 78 91 87 87 61 88 93 92 96 97 96 92 97 95 99 82 95 78 96 74 75 29 89 79 67 99 82 93 66 92 93 94 93 95 98 86 88 89 97 88 10 97 87 55

38 ± 4 13 ± 2 35 ± 1 37 ± 7 19 ± 2 5±2 14 ± 1 12 ± 2 4±2 0 0 5±3 10 ± 2 7±1 10 ± 1 4±2 7±1 14 ± 3 5±2 29 ± 5 19 ± 5 33 ± 1 42 ± 5 0 18 ± 2 13 ± 2 16 ± 3 0 8±2 10 ± 2 7±1 13 ± 3 8±3 39 ± 3 15 ± 5 30 ± 2 49 ± 4 19 ± 2 43 ± 16 21 ± 3 10 ± 1 9±1 11 ± 6 0 9±2 40 ± 2 16 ± 1 31 ± 4 63 ± 12 5±1

0 0 10 ± 8 9±8 19 ± 5 0 0 15 ± 2 0 0 0 0 0 0 0 0 0 0 0 0 10 ± 3 34 ± 2 0 15 ± 3 39 ± 7 27 ± 3 0 13 ± 4 28 ± 6 0 0 14 ± 5 24 ± 4 25 ± 3 0 0 0 29 ± 3 37 ± 6 20 ± 5 47 ± 19 7±4 42 ± 5 10 ± 4 18 ± 2 0 37 ± 6 49 ± 6 0 19 ± 8

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 82 84 86 88 90 92 94 96 98 100

78 94 94 95 78 89 53 91 93 93 15 87 44 88 41 85 87 84 93 62 94 40 90 97 20 96 93 88 89 94 95 92 91 96 97 99 63 91 44 90 81 99 72 96 98 70 82 18 90 91

23 ± 3 35 ± 5 23 ± 2 17 ± 1 5±1 6±4 10 ± 1 8±2 11 ± 4 9±3 0 40 ± 6 0 12 ± 4 19 ± 2 0 41 ± 10 27 ± 9 36 ± 6 19 ± 6 26 ± 1 26 ± 12 8±4 12 ± 1 10 ± 1 11 ± 2 7±1 4±1 35 ± 7 40 ± 1 8±3 23 ± 1 13 ± 2 43 ± 11 9±1 22 ± 4 5±2 24 ± 4 13 ± 6 0 0 22 ± 3 22 ± 4 0 0 0 10 ± 4 40 ± 7 38 ± 3 12 ± 1

0 0 22 ± 8 11 ± 2 0 0 0 0 0 0 43 ± 4 8±6 0 0 0 0 0 0 16 ± 6 0 9±4 6±3 29 ± 12 7±2 32 ± 8 28 ± 5 0 16 ± 2 0 24 ± 10 17 ± 4 0 0 0 70 ± 3 0 0 0 0 20 ± 12 0 17 ± 7 14 ± 5 20 ± 6 0 24 ± 8 34 ± 10 0 0 16 ± 2

The results are shown as the average percentage of the individual mean (± SD, standard deviation) for three experiments.

which are intermediates in flavonoid biosynthesis in plants, as a new promising scaffold for MtbPtps inhibition. In this work, we have synthesized and evaluated a library of 100 chalcone against both MtbPtpA and MtbPtpB. The results achieved led to the determination of potency and investigation of the structure− activity relationships (SAR), mechanism of inhibition, selectivity, and mode of interaction of this series. The integration of design, synthesis, biological evaluation, molecular

modeling, and SAR studies has proven useful for the identification of a new series of competitive and selective inhibitors of MtbPtpA and MtbPtpB.

2. RESULTS AND DISCUSSION 2.1. Chemistry. A chemical library of 100 chalcone derivatives was prepared by aldolic condensation of aromatic aldehydes and corresponding acetophenones (Schemes 1 and 393



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2), with yields ranging from 10 to 99% (Table 1). Compounds 1−42 are derived from 3,4-methylenedioxyacetophenone; compounds 43−47 are derived from 3,4-methylenedioxybenzaldehyde; compounds 48−84 are derived from 2-naphthylacetophenone; compounds 85−95 are derived from 2naphthaldehyde, while compounds 96−100 are derived from benzylated vanillin. All reagents used were commercially available, except benzylated vanillin (3-methoxy-4-(phenylmethoxy)-benzaldehyde) (prepared as previously described, with yield of 88%),34 2,4,5-trimethoxyacetophenone (synthesized as previously described with yield of 81%),35 and 2,4,6trimethoxyacetophenone (synthesized as previously described with yield of 85%).36 Compounds 43−47 and 85−95 were designed to complement the chalcone series previously assayed against MtbPtpA27 and compounds 96−100 to promote variations in the B-ring, maintaining promising substituents on the ring A. The strategy used for the synthesis of the other chalcones (1−42 and 48− 84) was the inversion of the rings of that previously published series.27 Because all the compounds have at least one aromatic ring, we selected the substituents first based on the Manual Method of Topliss,37a which comprises a fixed group of susbtituents to the aromatic mono- or disubstituted ring. Moreover, we expanded the series by the inclusion of other substituents and rings, promoting greater structural diversity. Still, it is noteworthy that all compounds were designed and fit in the “rule of five” (or Lipinski Rule).37b All synthesized compounds, including those previously reported by other authors (chalcones derivatives 1, 5−14, 19, 21, 25, 26, 28−30, 43, 45, 46, 50, 70, 79−81, 87, and 94)38 or by us (3, 16, 18, 23, 24, 27, 44, 47−49, 51, 53−61, 63, 65−69, 71, and 85)36b,39 were characterized by 1H NMR, 13C NMR, and IR (Supporting Information). The spectral characterization (1H NMR, 13C NMR, IR, and elementary analysis) of the novel compounds (2, 4, 15, 17, 20, 22, 31−42, 52, 62, 64, 72−78, 82−84, 86, 88−93, and 95−100) is detailed in the Experimental Section of the Supporting Information. 1H NMR spectra revealed that all structures are configured E (JHα−Hβ = ∼16 Hz). 2.2. Biochemical Evaluation and SAR Studies. The inhibitory activity of the chalcone derivatives against MtbPtpA and MtbPtpB was evaluated according to the standard method described elsewhere.27 The percentage of inhibition was assessed at a single concentration of 25 μM for each compound of the series (Table 1). As can be seen, several chalcone derivatives were active against both enzymes. In the case of MtbPtpA, the percentage of inhibition varied from 0 to 63%. Specifically, the results indicate that 25 out of 100 compounds (1, 4, 5, 7, 24, 34, 36, 38, 39, 42−45, 58, 60, 67, 68, 71, 73, 77, 91, and 95−98) exhibited enzyme inhibition (inhibition >25%). Regarding the MtbPtpB enzyme, 11 out of 100 compounds (22, 43, 49, 50, 70, 77, 81, 85, and 93−95) showed inhibition >30%. To better understand the molecular aspects involved in the inhibition of mycobacterium PTPs enzymes, we have determined IC50 values for all compounds presenting inhibitions higher than 25% and 30% for MtbPtpA and MtbPtpB, respectively (Table 2). The results indicate that compounds 5, 7, 39, 42−44, 58, 67, 68, 95, and 96 showed substantial inhibition of MtbPtpA with IC50 values 200 μM). The best inhibitory effect of MtbPtpA was achieved by compound 96 (IC50 = 15 μM), derived from benzylated vanillin structure, with methoxyl groups at positions 2 and 4 at the A-ring. In general, the 2naphthyl group as B-ring (compound 95) seems to favorably contribute to MtbPtpA inhibition. This observation is in good agreement with previous data that indicated significant π−π interactions between the 2-naphthyl substituent and the sidechain of the Trp48 amino acid residue of the MtbPtpA binding site.27,28 On the basis of the molecular information collected in the course of this work, the hydrophobicity of the B-ring as well as the presence of hydrogen bond donor/acceptor substituents in the A-ring seem to play an important role in the inhibitory activity of this series. Similar results were obtained for a series of chalcone-like derivatives evaluated against H37Rv Mtb strain.31 In that work, the most active compounds presented hydrophobic B-rings and polar-substituted A-rings. In the case of MtbPtpB, eight compounds (22, 43, 49, 50, 70, 85, 93, and 95) exhibited significant inhibition with IC50 values 100 45 ± 6 >100 >50 32 ± 4 15 ± 4

51 ± 8 >100 12 ± 2 25 ± 6 >50 >100

PTP1B IC50 (μM)

SI

± ± ± ± ± ±

0.5** 2.4* 258** 1.2** 3.1* 18*

23 107 3090 29 100 263

2 2 168 2 5 3

a The results are shown as the average of the individual mean ± SD. SI = selectivity index, given by *(IC50PTP1B/IC50MtbPtpA) or **(IC50PTP1B/ IC50MtbPtpB).

indexes obtained indicated that structural features play a key role on selectivity of this class of inhibitors. For example, compounds 44 and 95 exhibited moderate selectivity toward MtbPtpA (2.4- and 3.1-fold, respectively). In contrast, compound 96, which bears a 4-benzyloxy substituent in the B-ring, was highly selective toward MtbPtpA (about 18-fold). Regarding the MtbPtpB inhibitors, compound 43 showed a moderate shift toward the opposite way, being selective for the human enzyme, while compound 85 showed no selectivity. Conversely, compound 70, having a benzoic acid as B-ring, exhibited not only improved potency (IC50 = 12 μM) but also extraordinary 258-fold selectivity for MtbPtpB. With the goal of shedding light on the mechanism of inhibition of this series, these compounds were selected for further kinetic studies. 2.4. Kinetics Measurements and Mechanism of Inhibition. The definition of the mode of inhibition and binding relationships between the free enzyme (E), substrate (S), and enzyme−substrate (E•S) species to the inhibitor (I) allows the development of mechanism-based SARs, which are highly desirable for the rational design of enzyme inhibitors.41 To this end, we have determined the type of inhibition with respect to the substrate p-nitrophenyl phosphate (pNPP). Overall, the inhibitors exhibited degrees of saturation and release of p-nitrophenol that follows a classic Michaelis− Menten enzymatic mechanism.42 The Lineweaver−Burk double-reciprocal plots show intercepts of all lines (obtained at least with three different inhibitor concentrations) converging at the y-axis (1/Vmax), whereas the slope (Kmapp/ Vmax) and x-axis intercepts (−1/Kmapp) vary with inhibitor concentration (Figure 1). In these assays, the Vmax remains constant and the Kmapp values increase with increasing inhibitor concentrations. The behavior is consistent with a mutually exclusive binding mode, where the inhibitors compete with the substrate for the binding to the free enzyme active site.43 Therefore, the evaluated compounds are competitive inhibitors of MtbPtpA (compounds 44, 95, and 96) and MtbPtpB (compounds 43, 70, and 85). The Ki values obtained are in the low micromolar range (12−29 μM for MtbPtpA, and 8−13 μM for MtbPtpB) (Table 4), indicating that these inhibitors, 395



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Figure 1. Competitive inhibitory profile of compounds 96, 95, and 44 against MtbPtpA, and of compounds 70, 85, and 43 against MtbPtpB. Kinetic experiments were conducted in the presence of increasing concentrations of inhibitors: 0 μM (○), 5 μM (◆), 10 μM (▲), 15 μM (◇), 20 μM (□), 30 μM (●), 40 μM (△), 50 μM (■), 60 μM ( × ); pNPP was used as substrate in all experiments.

while the electron-rich 2-naphthyl group (B-ring) undergoes πstacking with the side-chain of the Arg166. Additionally, the nonpolar parts of compound 70 make favorable van der Waals contacts with several structural elements of the protein, some of them establishing hydrophobic interactions with the macromolecular counterparts, which significantly contribute to the complex stability. In particular, the aromatic ring of the benzoic acid substituent (A-ring) is in van der Waals contacts to the

side-chain of Met206 and the 2-naphthyl group (B-ring) is docked into the hydrophobic pocket formed by the amino acid residues Phe80, Pro81, and Phe133. The predicted interaction mode of compound 70 within the MtbPtpB binding site not only indicated the structural elements that might be related to the binding affinity but also provided insights into the molecular basis for selectivity. The protein tyrosine phosphatase family shares a catalytic domain 396



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Table 4. Ki Values of the MtbPtpA and MtbPtpB Inhibitorsa compd 43 44 70 85 95 96 a

MtbPtpA Ki (μM)

MtbPtpB Ki(μM)

IC50/Ki

13 ± 1

3.8 1.5 1.4 2.6 1.4 1.3

29 ± 1 8±1 10 ± 1 23 ± 1 12 ± 1

compound within the protein binding site as well as favorable nonpolar interactions that assist the stabilization of the inhibitor in the cavity. For the highly selective MtbPtB inhibitor 70, the proposed binding mode indicates that this compound occupies the narrow channel close to the catalytic Cys160, establishing attractive polar and hydrophobic contacts with the MtbPtpB active site residues. Their chemical versatility, activity, and selectivity suggest this class of competitive inhibitors as promising lead compounds for further medicinal chemistry efforts for the development of new anti-TB drugs.

Ki values are shown as the average of the individual mean ± SD.

containing a four-stranded parallel β-sheets that connects to a helix (helix α5) through a loop known as P loop. The P loop (residues 160−166) harbors the catalytic Cys160 within the invariant sequence HCX5R (Figure 3B).7 According to the proposed binding mode, the inhibitor is in favorable orientation to form van der Waals interactions with the side-chain of Phe161 (Figure 2B). The hydrophobic Phe161 residue is replaced with a polar Ser216 residue in the human homologue (Figure 3B). Therefore, the nonconservative substitution considerably changes the molecular recognition features of the binding sites. This finding is in good agreement with the experimental kinetic evaluation and might explain the high selectivity of the inhibitor 70 (∼260-fold) toward the mycobacterium enzyme (Table 3).

4. MATERIALS AND METHODS 4.1. Preparation of Compounds. Reagents used were obtained commercially (Sigma-Aldrich), except benzylated vanillin (3-methoxy4-(phenylmethoxy)-benzaldehyde), 2,4,5-trimethoxyacetophenone, and 2,4,6-trimethoxyacetophenone, which were synthesized as previously described.34−36 Chalcone derivatives (1−25 and 27−100) were prepared by aldolic condensation between acetophenones (1.0 mmol) and corresponding aldehydes (1.0 mmol), in methanol (15 mL), KOH (50% w/v), at room temperature with magnetic agitation for 24 h; the volume of KOH varied according to the reaction. When the aldehyde had photosensitive groups, the reactions were made in absence of light. Distilled water and 10% hydrochloric acid were added to the reaction for total precipitation of the compounds, which were then obtained by vacuum filtration and later recrystallized in dichloromethane and hexane. The purity of the synthesized compounds was analyzed by thin-layer chromatography (TLC) using Merck silica precoated aluminum plates of 200 μm thickness, with several solvent systems of different polarities. Compounds were visualized with ultraviolet light (λ = 254 and 360 nm) and using sulfuric anisaldehyde solution followed by heat application as the developing agent. Compound 26 was obtained by condensation between 3,4methylenedioxyacetophenone (1.0 mmol) and 3-methoxy-4-hydroxy-

3. CONCLUSIONS Our approach exploring the design, synthesis, and biological evaluation led to identification of a small series of chalcone derivatives as competitive inhibitors of MtbPtpA (44, 95, and 96) and MtbPtpB (43, 70, and 85). The predicted binding mode for the selective MtbPtpA inhibitor 96 shows polar interactions that play a key role in the orientation of this

Figure 2. (A) Predicted binding mode of compound 96 within the MtbPtpA active site. (B) Predicted binding mode of compound 70 within the MtbPtpB active site. Upper panel: inhibitors and protein residues involved in the ligand−receptor binding are indicated as ball-and-stick and stick models, respectively. Hydrogen bonds and π−π interactions are illustrated as blue and yellow dashed lines, respectively. Lower panel: view of the active site showing the surface charge distributions of MtbPtpA and MtPtpB. Positive and negative electrostatic potential (kT/e) are indicated in blue and red, respectively. This depiction was generated using the PyMOL APBS tools. 397



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Figure 3. Structure-based sequence alignment of (A) MtbPtpA and HCPTPA and (B) MtbPtpB and PTP1B. Conserved residues are highlighted in gray, and active site signature motifs of the PTP loop are shown in black boxes. The residues presumed to determine inhibitor selectivity that differ from those of human PTPs are shown in red boxes. The pairwise alignment was carried out with BLOSUM62 matrix implemented in BioEdit program. values for C), confirming ≥95% purity. The chemical data for each compound is presented as Supporting Information. 4.3. PTPs Expression and Purification. (a). PtpA wt and PtpB wt from Mycobacterium tuberculosis. PtpA and PtpB expression and purification were done as previously described,45 based in other described methodologies.46,47 (b). Human PTP1B wt. The plasmid pET19b encoding the 37 kDa form of the human wild type PTP1B (amino acid residues 1−321) was transformed into Escherichia coli BL21(DE3). Expression in E. coli BL21(DE3) was under the control of the T7 promoter. E. coli cells containing the recombinant plasmid were incubated overnight into 10 mL of LB broth containing 10 μL of ampicillin (100 mM) at 37 °C. Overnigth cultures of E. coli BL21(DE3) PTP1B (5 mL) were transferred to 250 mL of LB broth containing 250 μL of ampicillin (100 mM) and were grown with agitation (140 rpm) at 37 °C until an optical density of 0.85 at 600 nm was achieved, induced with IPTG (isopropyl β-D-thiogalactoside) to a final concentration of 0.6 mM. The cultures were grown for additional 20 h at room temperature and were harvested by centrifugation at 8000 rpm for 30 min. The next steps were carried out at 4 °C. The resulting pellet was resuspended in 40 mL of buffer A (20 mM imidazole pH 7.5, 1 mM EDTA, 3 mM DTT, and 10% glycerol) containing protease inhibitors (2 mM benzamidine and 2 μg/mL each of aprotinin, leupeptin, and pepstatin). The cells were lysed by sonication and spun down at 40000 rpm for 30 min. Purification was accomplished by a slight modification of the previously described method.48 The supernatant

benzaldehyde (1.0 mmol), in methanol (40 mL), KOH (50% v/v), at reflux for 4 h. Distilled water and 10% hydrochloric acid were added and the solution was left in the freezer for 24 h for total precipitation of the compound, which was obtained by vacuum filtration. The chalcone derivatives are soluble in dimethylsulfoxide, acetone, acetyl acetate, chloroform, and dichloromethane. Compounds 1, 5− 14, 19, 21, 25, 26, 28−30, 43, 45−47, 50, 70, 79−81, 87, and 94 were previously cited in the literature.38 Chalcones derivatives 3, 16, 18, 23, 24, 27, 44, 48, 49, 51, 53−61, 63, 65−69, 71, and 85 were published by our research group,36b,39 and 2, 4, 15, 17, 20, 22, 31−42, 52, 62, 64, 72−78, 82−84, 86, 88−93, and 95−100 are novel compounds. 4.2. Physicochemical Data of the Compounds. The structures were confirmed by melting points (mp), infrared spectroscopy (IR), and 1H and 13C nuclear magnetic resonance spectroscopy (NMR), as well as by elementary analysis for the previously undescribed ́ structures. Melting points were determined with a Microquimica MGAPF-301 apparatus and are uncorrected. IR spectra were recorded with an Abb Bomen FTLA 2000 spectrometer on KBr disks. NMR (1H and 13C) spectra were recorded on a Varian Oxford AS-400 (400 MHz) instrument, using tetramethylsilane as an internal standard; 1H NMR spectra revealed that all the structures were geometrically pure and configured E (JHα−Hβ = ∼16 Hz). Elementary analyses were carried out using a CHNS EA 1110; percentages of C and H were in agreement with the product formula (within ±0.4% of theoretical 398



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ligands within the MtbPtpA and MtbPtpB binding pockets. The X-ray crystallographic data for MtbPtpA determined at 1.9 Å (PDB ID 1U2P)5 and MtbPtpB determined at 2 Å (PDB ID 2OZ5)7 used in the docking simulations were retrieved from the Protein Data Bank (PDB). The ligands and water molecules from both structures were removed from the binding pockets. Hydrogen atoms were added in standard geometry using the Biopolymer module implemented in SYBYL 8.0. Histidines, glutamines, and asparagines residues within the binding site were manually checked for possible flipped orientation, protonation, and tautomeric states with Pymol 1.2 (DeLano Scientific, San Carlos, USA) side-chain wizard script. The binding site of MtbPtpA was defined as all the amino acid residues encompassed within a 10 Å radius sphere centered on the three-dimensional coordinates of the chloride ion bound to the active site.5 Similarly, the binding site of MtbPtpB was defined as all the amino acid residues encompassed within a 6 Å radius sphere centered on both the proximal and distal bound inhibitors.7 The docking procedures were repeated 30 times for each inhibitor. The GOLDScore scoring function and visual inspection were employed to select the representative conformation for each inhibitor.

was loaded on a 1 mL HiTrap Q FF column at 1.5 mL/min. The column was washed with buffer A until the absorbance at 280 nm was zero. The protein was eluted at 2 mL/min with a 100 mL linear gradient from zero to 0.5 M NaCl in buffer A. Fractions containing protein were pooled and desalted with a 35 mL HiPrep 26/10 column eluting with buffer B (20 mM bis−tris pH 6.5, 1 mM EDTA, 3 mM DTT, and 10% glycerol). Then, the resulting protein solution was loaded on a 1 mL HiTrap SP FF column at 1.5 mL/min. The column was washed with buffer B until the absorbance at 280 nm was zero. The protein was eluted at 2 mL/min with a 150 mL linear gradient from 0 to 0.5 M NaCl in buffer B. Protein concentrations were monitored by UV (A1 mg/mL280 nm = 1.00 mg/mL) and PTP1Bwt yield was 4.5 mg. The PTP1B fractions were stored at −80 °C. 4.4. Measurement of PtpA and PtpB Activity (%) and Inhibition (IC50). The phosphatase assays were carried out as previously described27 in 96-well plates containing 5 μL of diluted compounds at 1.0 × 10−3 M in DMSO (final concentration of 25 μM), 20 mM imidazol pH 7.0, 40 mM p-nitrophenyl phosphate [pNPP], and Milli-Q water to complete 198 μL in each well, followed by addition of 2 μL of recombinant protein in order to start the reaction. The proteins were used in the following concentrations: PtpA 1.0 μg/ μL, diluted 5 times in buffer D (20 mM Tris−HCl pH 8.0, 50 mM NaCl, 5 mM EDTA, 20% glycerol, and 5 mM DTT) and PtpB 0.4 μg/ μL, diluted 2 times in buffer D; both enzymes in a final concentration of 0.4 μg/μL. The enzymes, when active, cleave the substrate (pNPP), releasing p-nitrophenol, which have yellow color. The absorbance was measured by spectrophotometer UV−VIS (TECAN Magellan Infinite M200) for 10 min at 37 °C (at 410 nm with readings every 1 min). Negative controls were performed in the absence of enzyme and compound, and positive controls in the presence of enzyme and 100% DMSO. The percentage of residual activity was calculated as the difference in absorbance between the time 6 and 2 min, obtained by the average of two experiments carried out in triplicate. The IC50 values were determined with increasing concentrations of inhibitor (5−100 μM) versus % of inhibition, in triplicate in two independent experiments. The enzymatic activity was expressed in % of phosphatase residual activity compared with wells without inhibitor. The experimental data were analyzed with SigmaPlot Software 9.0 and the IC50 values determined by linear regression. It is important to stress the fact that all compounds are soluble in the assay mixtures at the described experimental conditions. 4.5. Selectivity Assay. The selectivity assays using PTP1B 2.0 μg/ μL, diluted 10 times in buffer B (20 mM bis−Tris pH 6.5, 1 mM EDTA, 3 mM DTT, 10% glycerol, and 92 mM NaCl) in a final concentration of 0.2 μg/μL, were carried out as described above. 4.6. Enzyme Kinectics. To determine the mecanism of inhibition, compounds 44, 95, and 96 were screened against PtpA and compounds 43, 70, and 85 against PtpB, by the same methodology described at section 4.4, however, varying concentrations of pNPP. The Ki values and the mechanism of inhibition of the compounds were determined using increasing concentrations of pNPP (0.5, 1.5, 3, 6, 8, 10, 15, 25 mM) for each concentration of compound (at least three concentrations ranging from 5 to 60 μM). The reaction rates were expressed as specific activity of the protein (μmol pNP min−1 mg−1) and the pNPP concentration in mM. The phosphatase released (1/V) was quantified and analyzed by the Lineweaver−Burk plot (1/[V] × 1/[S]) generated in the SigmaPlot Software 9.0. KMapp values obtained for each compound concentration were plotted versus [I], and the intercept of the curve at x-axis corresponding to −Ki. 4.7. Molecular Modeling. The 3D structures of the chalcone inhibitors were constructed using standard geometric parameters of the molecular modeling software package SYBYL 8.0. Each single optimized conformation of each molecule in the data set was energetically minimized employing the Tripos force field49 and the Powell conjugate gradient algorithm50 with a convergence criterion of 0.05 kcal/mol·Å and Gasteiger−Hückel charges.51 Molecular docking and scoring protocols were carried out with GOLD 4.1 (Cambridge Crystallographic Data Centre, Cambridge, UK).52 Default parameters were used to investigate the possible binding conformations of the

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ASSOCIATED CONTENT

S Supporting Information *

Chemical characterization of the compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*For H.T.: phone, +55 48 3721 6426; fax, +55 48 3721 9672; E-mail, [email protected]. For R.J.N.: phone, +55 48 3721 6844 r.236; fax, +55 48 3721 6850; E-mail, [email protected].

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ACKNOWLEDGMENTS We thank Prof. Dr. Pedro M. Alzari (Institut Pasteur, Paris) for the plasmids pRT28a (PtpA and PtpB) and Dr. Tiago A. S. Brandão (Universidade Federal de Minas Gerais) for the plasmid pET19b (PTP1B). We also thank the Departamento de Quı ́micaUniversidade Federal de Santa Catarina for the equipments for chemical analysis. We thank CNPq, CAPES, FAPESP, MCT, FAPESC, and FINEP for financial support and fellowships.

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ABBREVIATIONS USED BCG, Bacillus Calmette-Guérin; DMSO, dimethylsulfoxide; DTT, dithiothreitol; EDTA, ethylenediamine tetraacetic acid; ERK1/2, extracellular signal-regulated kinases 1 and 2; HCPTPA, human cytoplasmic protein tyrosine phosphatase; IFN-γ, interferon-γ; IL-6, interleukin 6; IPTG, isopropyl β-Dthiogalactoside; LMW PTP, low molecular weight protein tyrosine phosphatase; MDR, multidrug-resistance; Mtb, Mycobacterium tuberculosis; MtbPtps, Mycobacterium tuberculosis protein tyrosine phosphatases; MtbPtpA, Mycobacterium tuberculosis protein tyrosine phosphatase A; MtbPtpB, Mycobacterium tuberculosis protein tyrosine phosphatase B; OMTS, (oxalylamino-methylene)-thiophene sulfonamide; Ph, phenyl; pNPP, p-nitrophenyl phosphate; Ptks, protein tyrosine kinases; PtkA, protein tyrosine kinase A; PTP1B, human protein tyrosine phosphatase 1B; PTPs, protein tyrosine phosphatases; SAR, structure−activity relationships; TB, tuberculosis; TSP PTP, triple specificity protein tyrosine phosphatases; VPS33B, protein sorting 33 homologue B; WHO, World Health Organization 399



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Synthesis, Biological Evaluation, And Molecular Modeling of Chalcone

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